Substituted pyrimidines as vaccine adjuvants

ABSTRACT

The present invention provides a compound of the formula (2): 
     
       
         
         
             
             
         
       
         
         
           
             wherein X, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 8 , Y 1 , Y 2 , and L are as defined in the description, and a pharmaceutically acceptable salt thereof, which are useful as a vaccine adjuvant.

TECHNICAL FIELD

The present invention relates to a compound useful as a vaccineadjuvant, a method for producing the same, a composition comprising thesame, and a use of the same as a vaccine adjuvant.

BACKGROUND

Vaccines comprising a protein or a partial peptide, which are originatedfrom proteins or partial peptides produced by microorganisms, can beproduced using chemical synthesis or genetic recombination technologyand are advantageous in terms of safety and process for the production.On the other hand, however, subunit vaccines comprising a partialpeptide (epitope) tend to have lower immuno-stimulating ability thanthat of live vaccines and inactivated vaccines. Therefore, in order toenhance the immunogenicity of the epitope and to improve theimmuno-stimulating activity of the vaccine, it has been investigated forprophylactic or therapeutic methods using an adjuvant and an antigen incombination.

Adjuvants are an additive to enhance humoral and/or cellular immuneresponses to antigens, and Alum, saponin, etc. have been used as anvaccine adjuvant.

Recently, it was revealed that Toll-like Receptor (TLR) plays animportant role in the activation of innate immunity, which is a defensemechanism in living organisms against microorganisms, and thatmonophosphoryl lipid A (MPL), CPG ODN, etc., showed immuno-stimulatingeffect via TLR.

Of the known thirteen TLRs identified in human, five TLRs (TLRs 1, 2, 4,5 and 6) are involved in recognition of bacterial components, and fourTLRs (TLRs 3, 7, 8 and 9) are localized in cytoplasmic compartment andare involved in recognition of viral RNA (TLR 3, 7, 8) and unmethylatedDNA (TLR 9) (see Non Patent Document 1).

As an agonist (activator) of TLR7 and TLR8, small molecules that mimic asingle-stranded RNA of virus, which is a natural ligand, has been known.For example, synthetic compounds, such as pyrimidine compounds (PatentDocuments 1 and 2) and imidazoquinoline compounds (Patent Document 3),have been reported.

Activation of TLR7 and/or TLR8 with its agonist induces TH1 cells andactivates dendritic cells (DC) via TLR/MyD88-dependent signalingpathway. As a result, the expression of the T cell co-stimulatorymolecules (CD80, CD86, CD40) is enhanced, and inflammatory cytokinesincluding type I interferon (especially IFNα), TNFα, IL-6 or IL-12 areproduced.

In addition to the activation of DC, the TLR7 and/or TLR8 agonist(activator) was known to activate B cells and further stimulate NK cellsto promote IFNγ production, and therefore it is expected to have avaccine adjuvant activity. Indeed, adjuvant activity of TLR7/TLR8agonists, such as Resiquimod and Imiquimod, has been reported (NonPatent Document 2).

From the above, there is need for development of new vaccine adjuvantthat activates TLR7 and/or TLR8.

On the other hand, squalene is an oily substance used as an oilcomponent for oil-in-water and water-in-oil emulsion preparations, and asqualene-containing adjuvant such as MF59 has been used as an adjuvantfor influenza vaccine (Non Patent Documents 3, 4 and 5).

As for conjugates of TLR7/8 agonists and another substances, vaccineadjuvants wherein an imidazoquinoline compound are covalently linked toa fatty acid (Patent Documents 4, 5, 6 and Non-Patent Document 6),conjugates of an imidazoquinoline compound and a fatty acid glyceride(Patent Document 7), conjugates of an adenine compound and a fatty acidglyceride (Patent Document 8), and conjugates of an adenine compound anda phospholipid (Patent Document 9) were known. Also, conjugates whereinan adenine compound is conjugated with a fatty acid glyceride viapolyethylene glycol were known (Patent Document 10).

However, a conjugate of TLR7/8 agonist and squalene was not known.

PRIOR ART DOCUMENTS Patent Documents

-   [Patent Document 1] WO00/12487-   [Patent Document 2] WO2009/067081-   [Patent Document 3] U.S. Pat. No. 4,689,338-   [Patent Document 4] WO2005/001022-   [Patent Document 5] WO2005/018555-   [Patent Document 6] WO2012/024284-   [Patent Document 7] WO2010/048520-   [Patent Document 8] WO2011/017611-   [Patent Document 9] WO2011/139348-   [Patent Document 10] WO2010/093436

Non Patent Document

-   [Non Patent Document 1] Iwasaki, A., Nat. Immunol. 2004, 5, 987-   [Non Patent Document 2] Vaccine 2011, 29, 3341•M. A. Tomai et al,    Exp. Rev. Vaccine, 6, 835-   [Non Patent Document 3] G. Ott et al. Methods in Molecular Medicine,    2000, 42, 211-228-   [Non Patent Document 4] D. T. O'Hagan et al. Vaccine 2012, 4341-4348-   [Non Patent Document 5] C. B. Fox, molecules 2009, 14, 3286-   [Non Patent Document 6] Vaccine 2011, 29, 5434

SUMMARY OF INVENTION Problem to be Solved by the Invention

The present invention provides a compound useful as a vaccine adjuvant,a method for producing the same, and a composition comprising the same.

Solution to Problem

As a result of dedicated studies, the present inventors discovered thata conjugated compound, in which a pyrimidine compound which enhances thephysiological activity (function) of Toll-like Receptor 7 (TLR7) ischemically conjugated to an oily substance via a spacer, has an adjuvantactivity superior than that of the pyrimidine compound or the oilysubstance alone and have accomplished the present invention based onthis discovery.

The present invention is as set forth below.

[1] A compound of the formula (1) or a pharmaceutically acceptable saltthereof:

wherein

X is methylene, oxygen atom, sulfur atom, SO, SO₂ or NR⁷ wherein R⁷ ishydrogen atom or an alkyl group of 1 to 3 carbon atoms;

R¹ and R² are independently hydrogen atom or a substituted orunsubstituted alkyl group of 1 to 6 carbon atoms, provided that when thealkyl group is substituted, it is substituted with 1 to 4 same ordifferent substituents selected from the group consisting of hydroxygroup, halogen atom, and an alkoxy group of 1 to 6 carbon atoms;

R³ is an alkyl group of 1 to 6 carbon atoms, an alkoxy group of 1 to 6carbon atoms or an alkylthio group of 1 to 6 carbon atoms;

R⁴ is hydrogen atom, halogen atom, hydroxy group, an alkyl group of 1 to6 carbon atoms, an alkoxy group of 1 to 6 carbon atoms or cyano group;

Y¹ is a single bond, —(CR⁹R¹⁰)_(p)—, —CH═CH—(CR⁹R¹⁰)_(q)—, —C≡C—(CR⁹R¹⁰)_(q), — or —(CR⁹R¹⁰)_(r)—O—(CR^(9′)R^(10′))_(r′)— wherein R⁹,R¹⁰, R^(9′), and R^(10′) are independently hydrogen atom or an alkylgroup of 1 to 4 carbon atoms;

Y² is a single bond or —C(O)—;

L is a substituted or unsubstituted straight chain alkylene of 2 to 6carbon atoms or a divalent group derived from 4- to 8-memberedcycloalkane, provided that when the straight chain alkylene or thecycloalkane is substituted, it is substituted with 1 to 4 same ordifferent substituents selected from the group consisting of alkyl groupof 1 to 5 carbon atoms, hydroxy group and halogen atom, and any one ofthe methylene group in the straight chain alkylene of L is optionallyreplaced with carbonyl group;

R⁵ and R⁶ are independently hydrogen atom or a substituted orunsubstituted alkyl group of 1 to 6 carbon atoms, or either R⁵ or R⁶ isbonded to any carbon atom of L to form a 4- to 8-memberednitrogen-containing saturated heterocycle, or R⁵ and R⁶ are takentogether to form a substituted or unsubstituted 5- to 8-memberednitrogen-containing saturated heterocycle; provided that when the alkylgroup is substituted, it is substituted with same or different 1 to 4substituents selected from the group consisting of hydroxy group andhalogen atom; when the 4- to 8-membered nitrogen-containing saturatedheterocycle or the 5- to 8-membered nitrogen-containing saturatedheterocycle is substituted, it is substituted with same or different 1to 4 substituents selected from the group consisting of methyl group,ethyl group, propyl group, hydroxymethyl group, hydroxyethyl group,carbonyl group, hydroxy group and halogen atom;

m is 0 or 1;

p is an integer of 1 to 6;

q and q′ are independently an integer of 0 to 4;

r is an integer of 0 to 5, and r′ is an integer of 1 to 5, provided thatr′ is an integer of 2 or more when the sum of r and r′ is 5 or less andY² is a single bond; and

is independently a single bond or a double bond.

[2] The compound or a pharmaceutically acceptable salt thereof accordingto [1] wherein X is methylene, oxygen atom or NR⁷ wherein R⁷ is hydrogenatom or an alkyl group of 1 to 3 carbon atoms; R¹ and R² areindependently hydrogen atom or a substituted or unsubstituted alkylgroup of 1 to 6 carbon atoms, provided that when the alkyl group issubstituted, it is substituted with 1 to 4 same or differentsubstituents selected from the group consisting of hydroxy group andhalogen atom; R³ is an alkyl group of 1 to 6 carbon atoms; and R⁴ ishydrogen atom, halogen atom, hydroxy group, an alkyl group of 1 to 3carbon atoms or an alkoxy group of 1 to 3 carbon atoms.[3] The compound or a pharmaceutically acceptable salt thereof accordingto [1] or [2] wherein

are all single bonds or all double bonds.[4] The compound or a pharmaceutically acceptable salt thereof accordingto any one of [1] to [3] wherein Y¹ is a single bond or —(CR⁹R¹⁰)_(p)—wherein R⁹ and R¹⁰ are independently hydrogen atom or an alkyl group of1 to 4 carbon atoms; and Y² is a single bond or —C(O)—.[5] The compound or a pharmaceutically acceptable salt thereof accordingto [4] wherein Y¹ is a single bond; and Y² is —C(O)—.[6] The compound or a pharmaceutically acceptable salt thereof accordingto [4] wherein Y¹ is —(CR⁹R¹⁰)_(p)— wherein R⁹ and R¹⁰ are independentlyhydrogen atom or an alkyl group of 1 to 4 carbon atoms; p is an integerof 1 to 3; and Y² is a single bond.[7] The compound or a pharmaceutically acceptable salt thereof accordingto [3] wherein Y¹ is —(CR⁹R¹⁰)_(r)—O—(CR^(9′)R^(10′))_(r′)— wherein R⁹,R¹⁰, R^(9′) and R^(10′) are independently hydrogen atom or an alkylgroup of 1 to 4 carbon atoms; and r and r′ are independently 0, 1 or 2.[8] The compound or a pharmaceutically acceptable salt thereof accordingto any one of [1] to [7] wherein L is a straight chain alkylene of 2 or3 carbon atoms.[9] The compound or a pharmaceutically acceptable salt thereof accordingto [1]wherein

X is methylene;

R¹ is hydrogen atom or an alkyl group of 1 to 3 carbon atoms optionallysubstituted with hydroxyl group;

R² is hydrogen atom or an alkyl group of 1 to 6 carbon atoms;

R³ is an alkyl group of 1 to 3 carbon atoms;

R⁴ is hydrogen atom, halogen atom, hydroxy group, an alkyl group of 1 to3 carbon atoms or an alkoxy group of 1 to 3 carbon atoms;

Y¹ is a single bond or methylene;

Y² is a single bond or —C(O)—;

L is a straight chain alkylene of 2 or 3 carbon atoms;

R⁵ and R⁶ are independently hydrogen atom or an alkyl group of 1 to 3carbon atoms, or R⁵ and R⁶ are taken together to form a substituted orunsubstituted 5- to 8-membered nitrogen-containing saturatedheterocycle, provided that when the 5- to 8-membered nitrogen-containingsaturated heterocycle is substituted, it is substituted with same ordifferent 1 to 4 substituents selected from the group consisting ofhydroxy group and halogen atom;

m is 0 or 1; and

are all double bond.

[10] A pharmaceutical composition comprising the compound or apharmaceutically acceptable salt thereof according to any one of [1] to[9].

[11] The pharmaceutical composition according to [10] comprising furtheran antigen.

[12] The pharmaceutical composition according to [11] wherein theantigen is a pathogen-derived antigen or a tumor antigen.

[13] The pharmaceutical composition according to [11] or [12] whereinthe antigen is a peptide or a protein.

[14] A vaccine adjuvant comprising the compound or a pharmaceuticallyacceptable salt thereof according to any one of [1] to [9].

[15] The compound or a pharmaceutically acceptable salt thereofaccording to any one of [1] to [9] for use as a vaccine adjuvant.

[16] A method for enhancing an antigen-specific immune reaction in amammal, comprising administering the compound or a pharmaceuticallyacceptable salt thereof according to any one of [1] to [9] to themammal.

[17] A method for improving the immune response ability of a mammal,comprising administering the compound or a pharmaceutically acceptablesalt thereof according to any one of [1] to [9] to the mammal.

[18] Use of the compound or a pharmaceutically acceptable salt thereofaccording to any one of [1] to [9] for the manufacture of a vaccineadjuvant.

[19] A compound of the formula (2) or a pharmaceutically acceptable saltthereof:

wherein

X is methylene, oxygen atom, sulfur atom, SO, SO₂ or NR⁷ wherein R⁷ ishydrogen atom or an alkyl group of 1 to 3 carbon atoms;

R¹ and R² are independently hydrogen atom or a substituted orunsubstituted alkyl group of 1 to 6 carbon atoms, provided that when thealkyl group is substituted, it is substituted with 1 to 4 same ordifferent substituents selected from the group consisting of hydroxygroup, halogen atom, and an alkoxy group of 1 to 6 carbon atoms;

R³ is an alkyl group of 1 to 6 carbon atoms, an alkoxy group of 1 to 6carbon atoms or an alkylthio group of 1 to 6 carbon atoms;

-   R⁴ is hydrogen atom, halogen atom, hydroxy group, an alkyl group of    1 to 6 carbon atoms, an alkoxy group of 1 to 6 carbon atoms or cyano    group;

Y¹ is a single bond, —(CR⁹R¹⁰)_(p)—, —CH═CH—(CR⁹R¹⁰)_(q)—, —C≡C—(CR⁹R¹⁰)_(q′)— or —(CR⁹R¹⁰)_(r)—O—(CR^(9′)R^(10′))_(r′)— wherein R⁹,R¹⁰, R^(9′) and R^(10′) are independently hydrogen atom or an alkylgroup of 1 to 4 carbon atoms;

Y² is a single bond or —C(O)—;

L is a substituted or unsubstituted straight chain alkylene of 2 to 6carbon atoms or a divalent group derived from 4- to 8-memberedcycloalkane, provided that when the straight chain alkylene or thecycloalkane is substituted, it is substituted with 1 to 4 same ordifferent substituents selected from the group consisting of alkyl groupof 1 to 5 carbon atoms, hydroxy group and halogen atom, and any one ofthe methylene group in the straight chain alkylene of L is optionallyreplaced with carbonyl group;

R⁵ and R⁶ are independently hydrogen atom or a substituted orunsubstituted alkyl group of 1 to 6 carbon atoms, or either R⁵ or R⁶ isbonded to any carbon atom of L to form a 4- to 8-memberednitrogen-containing saturated heterocycle, or R⁵ and R⁶ are takentogether to form a substituted or unsubstituted 5- to 8-memberednitrogen-containing saturated heterocycle; provided that when the alkylgroup is substituted, it is substituted with same or different 1 to 4groups selected from the group consisting of hydroxy group and halogenatom; when the 4- to 8-membered nitrogen-containing saturatedheterocycle or the 5- to 8-membered nitrogen-containing saturatedheterocycle is substituted, it is substituted with same or different 1to 4 groups selected from the group consisting of methyl group, ethylgroup, propyl group, hydroxymethyl group, hydroxyethyl group, carbonylgroup, hydroxy group and halogen atom;

p is an integer of 1 to 6;

q and q′ are independently an integer of 0 to 4;

r is an integer of 0 to 5, and r′ is an integer of 1 to 5, provided thatr′ is an integer of 2 or more when the sum of r and r′ is 5 or less andY² is a single bond; and

R⁸ is hydrogen atom or an amino protecting group.

[20] The compound or a pharmaceutically acceptable salt thereofaccording to [19]

wherein

X is methylene;

R¹ is hydrogen atom or an alkyl group of 1 to 3 carbon atoms optionallysubstituted with hydroxy group;

R² is hydrogen atom or an alkyl group of 1 to 6 carbon atoms;

R³ is an alkyl group of 1 to 3 carbon atoms;

R⁴ is hydrogen atom, halogen atom, hydroxy group, an alkyl group of 1 to3 carbon atoms or an alkoxy group of 1 to 3 carbon atoms;

Y¹ is a single bond or methylene;

Y² is a single bond or —C(O)—;

L is a straight chain alkylene of 2 or 3 carbon atoms;

R⁵ and R⁶ are independently hydrogen atom or an alkyl group of 1 to 3carbon atoms, or R⁵ and R⁶ are taken together to form a substituted orunsubstituted 5- to 8-membered nitrogen-containing saturatedheterocycle; provided that when the 5- to 8-membered nitrogen-containingsaturated heterocycle is substituted, it is substituted with same ordifferent 1 to 4 groups selected from the group consisting of hydroxygroup and halogen atom.

Effect of the Invention

The present invention is able to provide an adjuvant that enhances aspecific immune reaction against an antigen useful as a vaccine.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the amount of OVA-specific IgG produced after immunizationwith OVA in mice which were administered intramuscularly with thecompounds of Examples 1 to 5.

FIG. 2 shows the results of ELISA for OVA-specific IgG1 and IgG2c inplasma of immunized mice which were administered intramuscularly withthe compounds of Example 1 and Example 4. The vertical axis representsOVA specific IgG2c/IgG1 ratio in plasma.

FIG. 3 shows the percentage of OVA-specific multifunctional CD4-positiveT lymphocytes in spleen cells of mice which were administeredintramuscularly with the compounds of Example 1 and Example 4.

FIG. 4 shows the percentage of MHC-restricted OVA-specific CD8-positiveT lymphocytes in spleen cells of mice which were administeredintramuscularly with the compounds of Example 1 and Example 4.

FIG. 5 shows the percentage of CD8-positive effector memory Tlymphocytes in spleen cells of mice which were administeredintramuscularly with the compounds of Example 1 and Example 4.

DESCRIPTION OF EMBODIMENTS

In case that a compound of the formula (I) as defined above exists inoptically active or racemic forms by virtue of one or more asymmetriccarbon atoms, the present disclosure includes in its scope any suchoptically active or racemic form having a physiological activity asreferred to hereinafter. The synthesis of such optically active formsmay be carried out by standard techniques of organic chemistry wellknown in the art, for example, a synthesis from optically activestarting materials or a resolution of a racemic mixture. Thephysiological activity may be evaluated using a standard laboratorytechnique as referred to hereinafter.

The compound of the formula (I) may exist in an unsolvated or solvatedform, such as a hydrate.

Also, the compound of the formula (1) may be deuterated form, whereinone or more ¹H are replaced with ²H(D).

The form of the compound of the formula (1) may be, but not limited to,amorphous or exist as a crystal. Crystal polymorphism may be present ina crystalline compound of the formula (1) or a pharmaceuticallyacceptable salt thereof, and thus, the compound of the present inventionincludes those in any crystal form.

The term “halogen atom” as used herein includes fluorine atom, chlorineatom, bromine atom, and iodine atom, and preferably, fluorine atom orchlorine atom.

The term “straight chain alkylene” as used herein includes a straightchain alkylene of 1 to 6 carbon atoms. Specific examples of the straightchain alkylene include, but are not limited to, methylene, ethylene,n-propylene and n-butylene.

The term “alkyl group” as used herein includes a straight or branchedchain alkyl group of 1 to 6 carbon atoms. Specific examples of the alkylgroups include, but are not limited to, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, and tert-butyl.

The term “cycloalkane” as used herein includes 4 to 8-memberedcycloalkanes, specifically, cyclobutane, cyclopentane, cyclohexane,cycloheptane and cyclooctane, and preferably, cyclopentane, cyclohexaneor cycloheptane.

The term “divalent group derived from cycloalkane” includes, but notlimited to, a divalent group capable of bonding to neighboring atoms ondifferent carbon atoms on the cycloalkane.

The term “alkoxy group” as used herein includes a straight or branchedchain alkoxy group of 1 to 6 carbon atoms. Specific examples of alkoxygroups include, but are not limited to, methoxy, ethoxy, propoxy,isopropoxyl, butoxy, isobutoxy, tert-butoxy, pentoxy, and isopentoxy.

The term “4- to 8-membered nitrogen-containing saturated heterocycle”includes 4- to 8-membered nitrogen-containing saturated heterocyclecontaining 1 to 3 hetero atoms selected from 2 or 3 nitrogen atoms, 0 or1 oxygen atom and 0 or 1 sulfur atom wherein at least two nitrogen atomsare contained in the ring. Specific examples include azetidine,pyrrolidine, piperidine, perhydroazepine, imidazolidine, piperazine,morpholine, thiomorpholine and perhydro-1,4-diazepine.

The “5- to 8-membered nitrogen-containing saturated heterocycle” may be5- to 8-membered ones of the aforementioned “4- to 8-memberednitrogen-containing saturated heterocycle”.

Examples of the substituent group for the nitrogen-containing saturatedheterocycle include preferably methyl group, ethyl group, propyl group,hydroxymethyl group, hydroxyethyl group, carbonyl group, hydroxy groupand halogen atom, and more preferably hydroxy group and halogen atom.The nitrogen-containing saturated heterocycle may be substituted withsame or different 1 to 4 said substituent groups.

In the formula (1), X represents methylene, oxygen atom, sulfur atom,SO, SO₂ or NR⁷ wherein R⁷ represents hydrogen atom or an alkyl group of1 to 3 carbon atoms. R⁷ preferably represents hydrogen atom or methylgroup. X preferably represents methylene.

In the formula (1), R¹ preferably represents hydrogen atom or an alkylgroup of 1 to 4 carbon atoms. Specific examples of R¹ include methylgroup, ethyl group, propyl group and butyl group.

In the formula (1), R² represents hydrogen atom or a substituted orunsubstituted alkyl group of 1 to 4 carbon atoms. When the alkyl groupis substituted, specific examples of the substituent group includehydroxy group. Specific examples of R² include hydrogen atom, methylgroup, ethyl group, propyl group, hydroxymethyl group and hydroxyethylgroup.

In the formula (1), R³ preferably represents an alkyl group of 1 to 4carbon atoms, an alkoxy group of 1 to 4 carbon atoms or an alkylthiogroup of 1 to 4 carbon atoms, more preferably an alkyl group of 1 to 3carbon atoms or an alkoxy group of 1 to 3 carbon atoms. Specificexamples of R³ include methyl group, ethyl group, propyl group or butylgroup, and more preferably methyl group.

In the formula (1), examples of R⁴ include preferably hydrogen atom, analkyl group of 1 to 3 carbon atoms, an alkoxy group of 1 to 3 carbonatoms, hydroxy or halogen atom, and more preferably hydrogen atom andmethoxy group.

In the formula (1), Y¹ preferably represents a single bond,—(CR⁹R¹⁰)_(p)— or —(CR⁹R¹⁰)_(r)—O—(CR^(9′)R^(10′))_(r′)— wherein R⁹,R¹⁰, R^(9′) and R^(10′) are independently hydrogen atom or an alkylgroup of 1 to 4 carbon atoms. R⁹, R¹⁰, R^(9′) and R^(10′) are preferablyindependently hydrogen atom or methyl group, more preferably hydrogenatom. More preferably, Y¹ is a single bond or —(CR⁹R¹⁰)_(p)—.

In the formula (1), when Y¹ is —(CR⁹R¹⁰)_(p)—, p is preferably aninteger of 1 to 4, more preferably 1, 2 or 3.

In the formula (1), when Y¹ is —CH═CH—(CR⁹R¹⁰)_(q)— or—C≡C—(CR⁹R¹⁰)_(q′)—, q and q′ are preferably independently an integer of0 to 3, more preferably 0 or 1.

In the formula (1), when Y¹ is —(CR⁹R¹⁰)_(r)—O—(CR^(9′)R¹⁰)_(r′)—, r ispreferably an integer of 0 to 3, r′ is preferably 1 to 4. The sum of rand r′ is 5 or less, more preferably r is 0 or 1, and r′ is 1 or 2.

In the formula (1), when Y¹ is a single bond or —(CR⁹R¹⁰)_(p)—, Y²represents a single bond or —C(O)-(carbonyl).

In a preferred embodiment, Y¹ represents a single bond and Y² represents—C(O)—.

In a preferred embodiment, Y¹ represents —(CR⁹R¹⁰)_(p)— and Y²represents a single bond wherein R⁹ and R¹⁰ are preferably independentlyhydrogen atom or methyl group, more preferably hydrogen atom, and p ispreferably an integer of 1 to 4, more preferably 1, 2 or 3.

According to one embodiment of the present invention, L in the formula(1) is a straight chain alkylene of 2 to 4 carbon atoms, preferablystraight chain alkylene of 2 or 3 carbon atoms, and more preferablyethylene.

In one embodiment of the present invention, L in the formula (1)represents a divalent group derived from 4- to 8-membered cycloalkane,more preferably a divalent group derived from 5- or 6-memberedcycloalkane. Specific examples of the divalent group include thefollowing divalent groups.

In one embodiment of the present invention, R⁵ and R⁶ in the formula (1)represents hydrogen atom or a substituted or unsubstituted alkyl groupof 1 to 3 carbon atoms, preferably independently hydrogen atom or analkyl group of 1 to 3 carbon atoms, more preferably independentlyhydrogen atom or methyl group, and still more preferably hydrogen atom.When the alkyl group is substituted, it is substituted with 1 to 4 sameor different substituents selected from hydroxy group or halogen atom.

In one embodiment of the present invention, either R⁵ or R⁶ in theformula (1) may be bonded to any carbon atom of L to form a 4- to8-membered nitrogen-containing saturated heterocycle, preferably a 4- to6-membered nitrogen-containing saturated heterocycle. Specific examplesof the 4- to 8-membered nitrogen-containing saturated heterocycle formedby R⁵ in combination with any carbon atom of L include the followings.

wherein R⁶ represents hydrogen atom or a substituted or unsubstitutedalkyl group of 1 to 6 carbon atoms.

Specific examples of the 4- to 8-membered nitrogen-containing saturatedheterocycle formed by R⁶ in combination with any carbon atom of Linclude the followings.

wherein R⁵ represents hydrogen atom or a substituted or unsubstitutedalkyl group of 1 to 6 carbon atoms.

In one embodiment of the present invention, R⁵ and R⁶ in the formula (1)may be taken together to form a substituted or unsubstituted 5- to8-membered nitrogen-containing saturated heterocycle, preferably 5- or6-membered nitrogen-containing saturated heterocycle. Specific examplesof such saturated nitrogen-containing hetero ring include thenitrogen-containing saturated heterocycles of the following formulae:

When either R⁵ or R⁶ is bonded to any carbon atom of L to form asubstituted 4- to 8-membered nitrogen-containing saturated heterocycleor when R⁵ and R⁶ are taken together to form a substituted 5- to8-membered nitrogen-containing saturated heterocycle, the substituentgroup are preferably same or different 1 to 4 substituents selected fromhydroxy group or a halogen atom.

In the formula (1), m preferably represents 1.

In the formula (1), the bonds represented by

independently are a single bond or a double bond, preferably all singlebonds or all double bonds, more preferably all double bonds.

In a preferred embodiment, the compound of the formula (1) is a compoundwherein

X represents methylene,

R¹ represents an alkyl group of 1 to 4 carbon atoms,

R² represents hydrogen atom, an alkyl group of 1 to 3 carbon atoms, oran alkyl group of 1 to 3 carbon atoms substituted with hydroxy group,

R³ represents an alkyl group of 1 to 3 carbon atoms or an alkoxy groupof 1 to 3 carbon atoms,

R⁴ represents hydrogen atom, halogen atom, hydroxy group, an alkyl groupof 1 to 6 carbon atoms or an alkoxy group of 1 to 6 carbon atoms,

Y¹ represents a single bond or —(CR⁹R¹⁰)_(p)—,

Y² represents —C(O)—,

L represents a straight chain alkylene of 2 to 6 carbon atoms,

R⁵ and R⁶ independently represent hydrogen atom or an alkyl group of 1to 3 carbon atoms or either R⁵ or R⁶ is bonded with any carbon atom of Lto form a 4- to 8-membered nitrogen-containing saturated heterocycle orR⁵ and R⁶ are taken together to form a substituted or unsubstituted 4-to 8-membered nitrogen-containing saturated heterocycle,

m represents 0 or 1, preferably 1,

represent all single bonds or all double bonds, or a pharmaceuticallyacceptable salt thereof.

In a preferred embodiment, the compound of the formula (1) is a compoundwherein

X represents methylene,

R¹ represents an alkyl group of 1 to 4 carbon atoms,

R² represents hydrogen atom, an alkyl group of 1 to 3 carbon atoms, oran alkyl group of 1 to 3 carbon atoms substituted with hydroxy group,

R³ represents an alkyl group of 1 to 3 carbon atoms or an alkoxy groupof 1 to 3 carbon atoms,

R⁴ represents hydrogen atom, halogen atom, hydroxy group, an alkyl groupof 1 to 6 carbon atoms or an alkoxy group of 1 to 6 carbon atoms,

Y¹ represents —(CR⁹R¹⁰)_(p)—

Y² represents a single bond,

L represents a straight chain alkylene of 2 to 6 carbon atoms,

R⁵ and R⁶ independently represent hydrogen atom or an alkyl group of 1to 3 carbon atoms or either R⁵ or R⁶ is bonded with any carbon atom of Lto form a 4- to 8-membered nitrogen-containing saturated heterocycle orR⁵ and R⁶ are taken together to form a substituted or unsubstituted 4-to 8-membered nitrogen-containing saturated heterocycle,

m represents 0 or 1, preferably 1,

represent all single bonds or all double bonds, or a pharmaceuticallyacceptable salt thereof.

In a preferred embodiment, the compound of the formula (1) is a compoundwherein

X represents methylene,

R¹ represents an alkyl group of 1 to 4 carbon atoms,

R² represents hydrogen atom, an alkyl group of 1 to 3 carbon atoms, oran alkyl group of 1 to 3 carbon atoms substituted with hydroxy group,

R³ represents an alkyl group of 1 to 3 carbon atoms or an alkoxy groupof 1 to 3 carbon atoms,

R⁴ represents hydrogen atom, halogen atom, hydroxy group, an alkyl groupof 1 to 6 carbon atoms or an alkoxy group of 1 to 6 carbon atoms,

Y¹ represents a single bond and Y² represents —C(O)—, or Y¹ represents—(CR⁹R¹⁰)_(p)— and Y² represents a single bond,

L represents a straight chain alkylene of 2 to 6 carbon atoms,

R⁵ and R⁶ independently represent hydrogen atom or an alkyl group of 1to 3 carbon atoms,

m represents 0 or 1, preferably 1,

represent all single bonds or all double bonds, or a pharmaceuticallyacceptable salt thereof.

Examples of preferred compounds of the present invention include thefollowing compounds or pharmaceutically acceptable salts thereof:

-   (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide;-   (4E,8E,12E,16E,20E)-1-(4-{4-[(2-amino-4-{[1-hydroxypentan-2-yl]amino}-6-methylpyrimidin-5-yl)methyl]-3-methoxybenzyl}piperazin-1-yl)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaen-1-one;-   (4E,8E,12E,16E,20E)-1-(4-{4-[(2-amino-4-{[1-hydroxypentan-2-yl]amino}-6-methylpyrimidin-5-yl)methyl]-3-methoxybenzoyl}piperazin-1-yl)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaen-1-one;-   4-[(2-amino-4-{[1-hydroxypentan-2-yl]amino}-6-methylpyrimidin-5-yl)methyl]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenoyl]amino}ethyl)-3-methoxybenzamide;    and-   4-[(2-amino-4-{[1-hydroxypentan-2-yl]amino}-6-methylpyrimidin-5-yl)methyl]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenoyl](methyl)amino}ethyl)-3-methoxybenzamide.

Alternatively,

-   (4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide;-   (4E,8E,12E,16E,20E)-1-(4-{4-[(2-amino-4-{[(2S)-1-hydroxypentan-2-yl]amino}-6-methylpyrimidin-5-yl)methyl]-3-methoxybenzyl}piperazin-1-yl)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaen-1-one;-   (4E,8E,12E,16E,20E)-1-(4-{4-[(2-amino-4-{[(2S)-1-hydroxypentan-2-yl]amino}-6-methylpyrimidin-5-yl)methyl]-3-methoxybenzoyl}piperazin-1-yl)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaen-1-one;-   4-[(2-amino-4-{[(2S)-1-hydroxypentan-2-yl]amino}-6-methylpyrimidin-5-yl)methyl]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenoyl]amino}ethyl)-3-methoxybenzamide;    and-   4-[(2-amino-4-{[(2S)-1-hydroxypentan-2-yl]amino}-6-methylpyrimidin-5-yl)methyl]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenoyl](methyl)amino}ethyl)-3-methoxybenzamide.

The present invention includes a composition comprising a plurality ofthe above compounds in combination.

Examples of the pharmaceutically acceptable salt of the compound of theformula (1) as used herein include acid addition salts or base additionsalts of the compound of the formula (1).

Examples of the acid addition salt include acid addition salts withinorganic or organic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, trifluoroacetic acid, citric acid and maleic acid.Examples of the base addition salt include alkali metal salts such assodium salt and potassium salt, alkaline earth metal salts such ascalcium salt, and ammonium salt.

The compound of the formula (1) can be produced by the followingprocesses using a known compound as a starting material.

The starting material may be used as a salt. The following processes aremerely illustrative, and the compound may be produced by another processappropriately based on the knowledge of one skilled in organicsynthesis.

[Process 1 for the Preparation of Compound (1)]

A compound of the formula (1) or a salt thereof can be prepared, forexample, by the following process.

Compound (1) can be prepared by reacting compound (3) with compound (2)in an inert solvent, using a condensing agent, optionally in thepresence of a base.

The base is not limited as long as it is used by a person skilled in theart in organic chemical reactions, and examples include organic basessuch as N-methyl morpholine, triethylamine, diisopropylethylamine,tributylamine, 1,8-diazabicyclo[4.3.0]non-5-ene,1,4-diazabicyclo[5.4.0]undec-7-ene, pyridine, dimethylaminopyridine andpicoline; and inorganic bases such as sodium bicarbonate, potassiumbicarbonate, sodium carbonate and potassium carbonate. The base may beused generally in an amount of 0.1 to 100 equivalents, preferably 1 to 5equivalents, to the compound (3).

The condensing agent may be those described in Experimental ChemistryCourse, Vol. 22, edited by The Chemical Society of Japan, Maruzen Co.,Ltd., and examples include phosphoric acid esters such as diethylcyanophosphate and diphenylphosphoryl azide; carbodiimides such as1-ethyl-3-(3-diethylaminopropyl)-carbodiimide hydrochloride (WSC•HCl)and dicyclohexylcarbodiimide (DCC); combinations of a disulfide such as2,2′-dipyridyl disulfide and a phosphine such as triphenylphosphine;phosphorus halides such as N,N′-bis(2-oxo-3-oxazolidinyl)phosphinicchloride (BOPC1); combinations of an azodicarboxylic acid diester suchas diethyl azodicarboxylate and a phosphine such as triphenylphosphine;2-halo-1-lower alkylpyridinium halides such as2-chloro-1-methylpyridinium iodide; 1,1′-carbonyldiimidazole (CDI);diphenylphosphoryl azide (DPPA); diethylphosphoryl cyanide (DEPC);tetrafluoroborates such as2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) and 2-chloro-1,3-dimethylimidazolidinium tetrafluoroborate (CIB);phosphates such as 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU),benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP), benzotriazol-1-yloxytris(pyrrolidino)phosphoniumhexafluorophosphate (PYBOP), and2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU).

Examples of the inert solvent include ether solvents such astetrahydrofuran, diethyl ether, 1,4-dioxane, 1,2-dimethoxyethane;hydrocarbon solvents such as hexane, heptane, toluene, benzene andxylene; halogenated hydrocarbon solvents such as dichloromethane,chloroform and dichloroethane; ketone solvents such as acetone; aproticsolvents such as acetonitrile, N,N′-dimethylformamide, dimethylsulfoxideand hexamethylphosphoramide; and a mixture thereof. The reactiontemperature is preferably selected from, but not limited to, the rangeof about −70° C. to 100° C., more preferably 0° C. to 40° C.

Alternatively, Compound (3) may be converted to an acid halide using ahalogenating agent (e.g., 1-chloro-N,N,2-trimethylpropenylamine,phosphorus oxychloride, phosphorus trichloride, thionyl chloride,phosphorus pentachloride) and then reacted with Compound (2) in an inertsolvent, optionally in the presence of a base, to obtain Compound (1).

Alternatively, Compound (3) may be converted to an acid halide using ahalogenating agent (e.g., 1-chloro-N,N,2-trimethylpropenylamine,phosphorus oxychloride, phosphorus trichloride, thionyl chloride,phosphorus pentachloride) and then reacted with Compound (2) in an inertsolvent, optionally in the presence of a base, to obtain Compound (1).

Examples of the inert solvent include ether solvents such astetrahydrofuran, diethyl ether, 1,4-dioxane, 1,2-dimethoxyethane;hydrocarbon solvents such as hexane, heptane, toluene, benzene andxylene; halogenated hydrocarbon solvents such as dichloromethane,chloroform and dichloroethane; ketone solvents such as methylethylketone and acetone; and aprotic solvents such as acetonitrile,N,N′-dimethylformamide, dimethylsulfoxide and hexamethylphosphoramide.Examples of the base include organic bases such as N-methyl morpholine,triethylamine, diisopropylethylamine, tributylamine,1,8-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[5.4.0]undec-7-ene,pyridine, dimethylaminopyridine, picoline. The halogenating agent can beused in an amount of 0.1 to 100 equivalents, preferably 0.8 to 3equivalents, to the compound (3). The reaction temperature is preferablyselected from, but not limited to, the range of about −30° C. to 60° C.

Compound (3) can be prepared according to a method well known in the art(see Org. Biomol. Chem. 2011, 9, 4367).

[Process 1 for the Preparation of Compound (2)]

Compound (2a), wherein Y² in the formula (2) is —C(O)—, or a saltthereof can be prepared, for example, by the following process.

wherein X, R¹, R², R³, R⁴, R⁵, R⁶, yl and L are as defined above.

Compound (2a) can be synthesized from Compound (4) and Compound (5a)according to a method as described for the preparation of Compound (1).

Compound (4) can be prepared according to a method well known in theart, such as those described in WO 2009/067081 and WO 2010/103885.

[Process 2 for the Preparation of Compound (2)]

Compound (2b), wherein Y² in the formula (2) is a single bond, or a saltthereof can be prepared, for example, by the following process.

wherein X, R¹, R², R³, R⁴, R⁵, R⁶ and L are as defined above,Y¹ is —(CR⁹R¹⁰)_(p)—, —CH═CH—(CR⁹R¹⁰)_(q)—, or—C≡C—(CR⁹R¹⁰)q′-wherein p, q and q′ are as defined above, and(1) Q¹ represents —Y¹NHR⁵ and Q² represents CHO; or(2) Q¹ represents —Y^(1′)—CHO, wherein Y^(1′) is absent or representsalkylene and —Y^(1′)—(CR⁹R¹⁰)— corresponds to —Y¹—, and Q² represents—CH₂NHR⁵.

Compound (2b) can be prepared by coupling Compound (6) and the compound(5b) under a condition for reductive amination well known in the art.Specifically, an aldehyde compound and an amine compound can be reactedin a solvent with a reducing agent, such as sodium triacetoxyborohydrideand sodium cyanoborohydride, in the presence or absence of an acid suchas acetic acid to prepare Compound (2b).

Compound (6) can be prepared according to a method well known in theart, such as those described in WO2010/133885, WO2012/066335, andWO2012/066336.

[Process 3 for the Preparation of Compound (2)]

Also, Compound (2b) or a salt thereof can be prepared according to thefollowing process.

wherein X, R¹, R², R³, R⁴, R⁵, R⁶ and L are as defied above, and Y¹ is—(CR⁹R¹⁰)_(p)—, —CH═CH—(CR⁹R¹⁰)_(q)—,—C≡C—(CR⁹R¹⁰)q′-, or—(CR⁹R¹⁰)_(r)—O—(CR^(9′)R^(10′))_(r′)—, wherein R⁹, R¹⁰, R^(9′),R^(10′), p, q, q′, r and r′ are as defined above, and LG is a leavinggroup.Step 1

Compound (7) can be prepared according to a method well known in the art(see WO 2010/133885, WO 2012/066336, etc.). The leaving group LG inCompound (8) is not limited so long as it is well known in the art, anda halogen atom, an alkylsulfonyloxy group, an arylsulfonyloxy group, orthe like can be used appropriately.

Compound (8) can be prepared by reacting Compound (7) withmethanesulfonyl chloride, p-toluenesulfonyl chloride or the like, in thepresence of a base such as triethylamine, diisopropylethylamine,pyridine, dimethylaminopyridine, sodium carbonate, potassium carbonateor the like. The reaction temperature is preferably selected from, butnot limited to, the range of about 0° C. to 120° C.

Step 2

Compound (2b) can be prepared by reacting Compound (8) with Compound(5a) in an inert solvent in the presence of a base.

The base is not limited so long as it is used as a base in a usualreaction, and examples include organic bases such as N-methylmorpholine, triethylamine, diisopropylethylamine, tributylamine,1,8-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[5.4.0]undec-7-ene,pyridine, dimethylaminopyridine; inorganic bases such as sodiumbicarbonate, potassium bicarbonate, sodium carbonate, potassiumcarbonate, cesium carbonate, sodium hydroxide and sodium hydride. Thebase may be used generally in an amount of 0.1 to 100 equivalents,preferably 1 to 3 equivalents, to Compound (8).

Examples of the inert solvent include ether solvents such astetrahydrofuran, diethyl ether, 1,4-dioxane, 1,2-dimethoxyethane;hydrocarbon solvents such as hexane, heptane, toluene, benzene andxylene; aprotic solvents such as acetonitrile, N,N′-dimethylformamide,N-methyl pyrrolidone, dimethylsulfoxide; and a mixture thereof. Thereaction temperature is preferably selected from, but not limited to,the range of about 0° C. to 120° C.

[Process 4 for the Preparation of Compound (2)]

Also, Compound (2b) or a salt thereof can be prepared according to thefollowing process.

wherein X, R¹, R², R³, R⁴, R⁵ and R⁶ are as defined above, L issubstituted or unsubstituted alkylene of 2 to 6 carbon atoms, Y¹ is asingle bond, —(CR⁹R¹⁰)_(q)—, —CH═CH—(CR⁹R¹⁰)_(q)—, —C≡C—(CR⁹R¹⁰)q′-,or —(CR⁹R¹⁰)_(r)—O—(CR^(9′)R^(10′))_(r′)—, wherein R⁹, R¹⁰, R^(9′),R^(10′), q, q′, r and r′ are as defined above, and LG is a leavinggroup.

Compound (2b) can be prepared in the similar manner as described in Step2 of Process 3 for the preparation of Compound (2), using Compound (9)and Compound (5c).

Compound (9) can be prepared according to a method well known in theart, such as those described in WO2010/133885, and WO2012/066336.

[Process 5 for the Preparation of Compound (2)]

Compound (2c) or a salt thereof can be prepared according to thefollowing process.

wherein X, R¹, R², R³, R⁴, R⁵ and R⁶ are as defined above, L′ issubstituted or unsubstituted alkylene of 1 to 5 carbon atoms, Y¹ is asingle bond, —(CR⁹R¹⁰)_(p)—, —CH═CH—(CR⁹R¹⁰)_(q)—, —C≡C—(CR⁹R¹⁰)q′-, or—(CR⁹R¹⁰)_(r)—O—(CR^(9′)R^(10′))_(r′)— wherein R⁹, R¹⁰, R^(9′), R^(10′),p, q, r and r′ are as defined above.

Compound (2c) can be prepared as described in the process for thepreparation of Compound (1), using Compound (9) and Compound (5d).

The present invention provides intermediates in the processes describedabove. Examples of such intermediates include compounds of the formula(2):

wherein

X is methylene, oxygen atom, sulfur atom, SO, SO₂ or NR⁷ wherein R⁷ ishydrogen atom or an alkyl group of 1 to 3 carbon atoms;

R¹ and R² are independently hydrogen atom or a substituted orunsubstituted alkyl group of 1 to 6 carbon atoms, provided that when thealkyl group is substituted, it is substituted with 1 to 4 same ordifferent substituents selected from the group consisting of hydroxygroup, halogen atom, and an alkoxy group of 1 to 6 carbon atoms;

R³ is an alkyl group of 1 to 6 carbon atoms, an alkoxy group of 1 to 6carbon atoms or an alkylthio group of 1 to 6 carbon atoms;

R⁴ is hydrogen atom, halogen atom, hydroxy group, an alkyl group of 1 to6 carbon atoms, an alkoxy group of 1 to 6 carbon atoms or cyano group;

Y¹ is a single bond, —(CR⁹R¹⁰)_(p)—, —CH═CH—(CR⁹R¹⁰)_(q)—,—C≡C—(CR⁹R¹⁰)_(q′)—, or

—(CR⁹R¹⁰)_(r)—O—(CR^(9′)R^(10′))_(r′)— wherein R⁹, R¹⁰, R^(9′), andR^(10′) are independently hydrogen atom or an alkyl group of 1 to 4carbon atoms;

Y² is a single bond or —C(O)—;

L is a substituted or unsubstituted straight chain alkylene of 2 to 6carbon atoms or a divalent group derived from 4- to 8-memberedcycloalkane, provided that when the straight chain alkylene or thecycloalkane is substituted, it is substituted with 1 to 4 same ordifferent substituents selected from the group consisting of alkyl groupof 1 to 5 carbon atoms, hydroxy group and halogen atom, and any one ofthe methylene group in the straight chain alkylene of L is optionallyreplaced with carbonyl group;

R⁵ and R⁶ are independently hydrogen atom or a substituted orunsubstituted alkyl group of 1 to 6 carbon atoms, or either R⁵ or R⁶ isbonded to any carbon atom of L to form a 4- to 8-memberednitrogen-containing saturated heterocycle, or R⁵ and R⁶ are takentogether to form a substituted or unsubstituted 5- to 8-memberednitrogen-containing saturated heterocycle; provided that when the alkylgroup is substituted, it is substituted with same or different 1 to 4groups selected from the group consisting of hydroxy group and halogenatom; when the 4- to 8-membered nitrogen-containing saturatedheterocycle or the 5- to 8-membered nitrogen-containing saturatedheterocycle is substituted, it is substituted with same or different 1to 4 groups selected from the group consisting of methyl group, ethylgroup, propyl group, hydroxymethyl group, hydroxyethyl group, carbonylgroup, hydroxy group and halogen atom;

p is an integer of 1 to 6;

q and q′ are independently an integer of 0 to 4;

r is an integer of 0 to 5, and r′ is an integer of 1 to 5, provided thatr′ is an integer of 2 or more when the sum of r and r′ is 5 or less andY² is a single bond; and

R⁸ is hydrogen atom or an amino protecting group, and pharmaceuticallyacceptable salts thereof.

The protecting group of R⁸ in the formula (2) may be, but not limitedto, a group commonly used as a protecting group for amino group.Specific examples include carbamates such as t-butoxycarbonyl group,benzyloxycarbonyl group and 9-fluorenylmethyloxycarbonyl group; benzylgroup; sulfones such as nosyl group; imides such as phthalimide.

In a process of the invention, if a specific functional group, such ashydroxy group or amino group, in a reagent is necessary to be protected,protection/deprotection may be conducted with one or more protectinggroup at an appropriate step in the process according to a procedurewell known in the art.

The protection/deprotection of functional groups is described in J. W.F. McOmie, Ed., “Protective Groups in Organic Chemistry”, Plenum Press(1973) and T. W. Greene and P. G. M. Wuts, “Protective Groups in OrganicSynthesis,” 3rd Edition, Wiley-Interscience (1999).

The present invention provides a pharmaceutical composition comprising acompound of the formula (1) or a pharmaceutically acceptable saltthereof as defined above, in combination with a pharmaceuticallyacceptable diluent or carrier.

The pharmaceutical composition can be used as an adjuvant formaintaining or enhancing the immunostimulatory activity of the activeingredient having immunostimulatory activity.

That is, the compound of the present invention or a pharmaceuticallyacceptable salt thereof has an activity of inducing or enhancing anantigen-specific antibody, specifically an antigen-specific IgG, morespecifically a TH1 type antigen-specific IgG (e.g., IgG2c).

In addition, the compound of the present invention or a pharmaceuticallyacceptable salt thereof has an activity of inducing or enhancingCD4-positive (i.e., MHC class 2-restricted) and/or CD8-positive (i.e.,MHC Class 1-restricted) T lymphocytes.

Furthermore, the compound of the present invention or a pharmaceuticallyacceptable salt thereof has an activity of inducing or enhancingMHC-restricted antigen-specific T lymphocytes.

Also, the compound of the present invention or a pharmaceuticallyacceptable salt thereof has an activity of inducing or enhancing memoryT lymphocytes, specifically CD8-positive effector memory T lymphocytes.

In addition, the compound of the present invention or a pharmaceuticallyacceptable salt thereof, when it is administered to a mammal, ischaracterized, in that the activity to induce systemic inflammatorymediators, i.e., to raise the concentration of interferon-α,interferon-γ, IL-6, IP-10, or the like, is lower than that of the samedose of the compound without squalene structure.

The pharmaceutical composition may contain an antigen. Examples of theantigen include a tumor antigen protein; a tumor antigen peptide derivedfrom said tumor antigen protein, such as NY-ESO-1, MAGE-3, WT1 andHer2/neu; a hypervariable region of an antibody; and a pathogen-derivedantigen such as a protein or a partial peptide thereof derived from avirus or a bacterium. Also, a complex of such antigen and a carrier isincluded in the scope of the antigen as used herein. Examples of suchcomplex include those wherein an antigen (including, but not limited to,proteins and peptides) are chemically bonded to a protein that serves asa carrier via a linker well known in the art; and those wherein anantigen is contained in a virus-like particle (VLP). Therefore, thecompound of the present invention or a pharmaceutically acceptable saltthereof, by using in combination with the above-mentioned antigen, isuseful as a drug for the treatment or prevention of cancer, infectionwith virus or bacteria.

Also, the compound of the present invention or a pharmaceuticallyacceptable salt thereof can be used as an adjuvant to assistimmunostimulation in a treatment for inducing other immunological orimmune reaction. Specific Examples of the treatment include ex vivo andin vivo approaches to enhance the immunogenicity of tumor cells of apatient (e.g., transfection with cytokines such as interleukin 2,interleukin 4 or granulocyte macrophage colony stimulating factor),approaches to reduce T cell anergy, approaches using transfected immunecells (e.g., cytokine-transfected dendritic cells), approaches usingcytokine-transfected tumor cell lines, approaches to reduce the functionof immunosuppressive cells (e.g., regulatory T cells, bonemarrow-derived repressed cells and IDO (indoleamine2,3-dioxygenase)-expressing dendritic cells), and radiation therapy toinduce an immune response.

Examples of the administration route of the pharmaceutical compositionincludes parenteral administration, specifically intravascular (e.g.,intravenous), subcutaneous, intradermal, intramuscular, intranasal, andpercutaneous administrations.

In one embodiment, the pharmaceutical composition of the presentinvention may contain a compound of the formula (1) or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable diluent or carrier.

The dosage form of the pharmaceutical composition of the presentinvention may be a liquid formulation for injection or nasal drops or afreeze-dried formulation prepared by lyophilizing the liquidformulation.

Examples of the liquid formulation for injection include an emulsioncontaining an aqueous solution and an oily composition, a liposome, anaqueous solution or suspension wherein an active ingredient and/or acompound of the formula (1) or a pharmaceutically acceptable saltthereof are dissolved or dispersed in water, and an oily solution orsuspension wherein an active ingredient and/or a compound of the formula(1) or a pharmaceutically acceptable salt thereof are dissolved ordispersed in oil.

Examples of the liquid formulation for nasal drops include an emulsioncontaining an aqueous solution and an oily composition, a liposome, anaqueous solution or suspension wherein an active ingredient and/or acompound of the formula (1) or a pharmaceutically acceptable saltthereof are dissolved or dispersed in water.

The aqueous solution, aqueous solution formulation or aqueous suspensionformulation may be an aqueous solution or aqueous suspension containingdistilled water for injection, and appropriately, a buffer, a pHadjusting agent, a stabilizer, an isotonizing agent or an emulsifyingagent.

The oily composition, oily solution formulation or oily suspensionformulation may be a composition containing a vegetable oil and fat,animal oil and fat, a hydrocarbon, a fatty acid ester, or the like, morespecifically, a composition containing squalene, squalane, or the like.

The emulsion as used herein may be an oil-in-water emulsion or awater-in-oil emulsion. The oil as used in the oil-in-water emulsion isnot limited so long as it is capable of dissolving or uniformlydispersing the compound of the formula (1) (or a pharmaceuticallyacceptable salt thereof), but may be a vegetable oil and fat, an animaloil and fat, a hydrocarbon, a fatty acid ester, or the like, and morespecifically, squalene, squalane, or the like. The oil as used in thewater-in-oil emulsion is not limited so long as it is capable ofdissolving or uniformly dispersing the compound of the formula (1) (or apharmaceutically acceptable salt thereof), but may be a vegetable oiland fat, an animal oil and fat, a hydrocarbon, a fatty acid ester, orthe like.

Specifically, the oil-in-water emulsion may contain 0.1 to 10 wt % ofsqualene. In one embodiment, the oil-in-water emulsion may contain 0.001to 1 wt % of a compound of the formula (1) or a pharmaceuticallyacceptable salt thereof and 1 to 10 wt % of squalene. The oil-in-wateremulsion may further comprise one or more surfactants or bufferingagents. The surfactant and its amount are not limited so long as theyare preferable to maintain a uniform emulsified state, and examplesinclude polysorbates, polyoxyethylene hydrogenated castor oils,polyoxyethylene polyoxypropylene glycols, fatty acid esters ofpolyalcohols and fatty acid esters, for example, 0.1 to 5 wt % ofpolysorbate 80, 0.1 to 5 wt % of sorbitan trioleate (e.g., Span 85™).Examples of the buffering agent include phosphate and organic acid salt.

In preparation of a freeze-dried formulation, an excipient may be addedas appropriate. The excipient and its amount are not limited so long asthey are preferable to form good freeze-dried cake or lyophilizedpowder, and examples include saccharides, sugar alcohols, amino acidsand sodium chloride, for example, 0.1 to 20% mannitol.

The pharmaceutical composition of the present invention may furthercontain other additives, and examples of such additives includesurfactants, antioxidants, preservatives, and soothing agents.

The compound of the formula (1) or a pharmaceutically acceptable saltthereof may be administered simultaneously with or at any intervalbefore or after the antigenic substance (immunogen) in a unit doseranging from generally 5 to 5000 mg/m² of body surface area, i.e., about0.1 ng/kg to 100 mg/kg, which provides a prophylactically ortherapeutically effective dose. The unit dosage form for injections,tablets or capsules generally contains, for example, 1 ng to 250 mg ofactive ingredient, and preferably, used at a dose ranging from 1 ng to50 mg/kg per day. However, the daily dose may vary depending on the hostto be treated, the route of administration and the severity of thedisease being treated. Thus, the optimal dose can be determined by apractitioner who treats individual patient or warm-blooded animal.

The term “treatment” as used herein means alleviating some or all of thesymptoms of disease, in whole or in part, or preventing or delaying theprogression of disease.

The term “prevention” as used herein means primary prevention of disease(prevention of onset of disease) or secondary prevention (prevention ofrelapse in a patient whose symptom has been alleviated or disease hasbeen cured after the onset of the disease, prevention of recurrence).

Since the compound of the present invention or its pharmaceuticallyacceptable salt has an immune adjuvant activity in vitro or in vivo, itis useful as a vaccine adjuvant for maintaining or enhancing theimmunogenicity of the antigen.

The compound of the present invention or a pharmaceutically acceptablesalt thereof can be used for maintaining or enhancing the effect of animmuno-stimulating substance (immunostimulatory substance) which is anagent for treating or preventing a disease, i.e., a substance inducingan antigen-specific immune reaction.

A pharmaceutical composition comprising a compound of the presentinvention or a pharmaceutically acceptable salt thereof and a substanceenhancing an antigen-specific immune reaction (also referred to as anantigen) is also an embodiment of the present invention. The antigen maybe, but not limited to, an antigen protein, an antigen peptide (partialpeptide) derived from said antigen protein, or a complex thereof with acarrier.

In a specific embodiment of the invention, a compound of the inventionor a pharmaceutically acceptable salt thereof can be administered incombination with a tumor antigen protein or a tumor antigen peptide forcancer immunotherapy to treat or prevent cancer. Examples of the cancerinclude common cancers such as bladder cancer, head and neck cancer,prostate cancer, breast cancer, lung cancer, ovarian cancer, pancreaticcancer, cancer of intestine and colon, stomach cancer, skin cancer andbrain cancer; malignant diseases (Hodgkin's lymphoma and non-Hodgkin'slymphoma, etc.) that affect bone marrow (including leukemia) andlymphoproliferative system. The treatment or prevention of cancer asused herein may be prevention of metastatic disease and tumorrecurrence, and prevention and treatment of paraneoplastic syndrome.

Examples of the antigen include, but not limited to, MAGE (Science, 254:p 1643 (1991)), gp 100 (J. Exp. Med., 179: p 1005 (1994)), MART-1 (Proc.Natl. Acad. Sci. USA, 91: p3515 (1994)), tyrosinase (J. Exp. Med., 178:p489 (1993)), MAGE related proteins (J. Exp. Med., 179: p921 (1994)),β-catenin (J. Exp. Med., 183: p1185 (1996)), CDK4 (Science, 269: p1281(1995)), HER2/neu (J. Exp. Med., 181: p2109 (1995)), mutant p53 (Proc.Natl. Acad. Sci. USA, 93: p14704 (1996)), CEA (J. Natl. Cancer. Inst.,87: p982 (1995)), PSA (J. Natl. Cancer. Inst., 89: p293 (1997)), WT1(Proc. Natl. Acad. Sci. USA, 101: p13885 (2004)), HPV-derived antigen(J. Immunol., 154: p5934 (1995)), and EBV-derived antigen (Int.Immunol., 7: p653 (1995)).

Examples of the tumor antigen peptide derived from the cancer antigeninclude, but not limited to, MAGEA3 peptide 168-176 (Coulie P G et al.,Immunol. Rev. 188: 33 (2002)), gp100 peptide 209-217 (Rosenberg S A etal., Nat. Med. 4: 321 (1998)), gp100 peptide 280-288 (Phan G Q et al.,Proc. Natl. Acad. Sci. USA 100: 8372 (2003)), Melan-A peptide 27-35(Cormier J N et al., Cancer J. Sci. Am. 3: 37 (1997)), Melan-A peptide26-35, Tyrosinase peptide 1-9, Tyrosinase peptide 368-376, gp100 peptide280-288, gp100 peptide 457-466 (Jager E et al., Int. J. Cancer 67: 54(1996)), HER-2 peptide 369-384, HER-2 peptide 688-703, HER-2 peptide971-984 (Knutson K L et al., J. Clin. Invest. 107: 477 (2001)), andMAGE-A12 peptide 170-178 (Bettinotti M P et al., Int. J. Cancer 105: 210(2003)).

In addition, the compound of the present invention or itspharmaceutically acceptable salt, by administering in combination withan active ingredient of a vaccine for preventing infectious diseases,can prevent various infectious diseases such as genital wart, commonwart, plantar wart, hepatitis B, hepatitis C, herpes simplex virus,molluscum contagiosum, smallpox, human immunodeficiency virus (HIV),human papilloma virus (HPV), RS virus, norovirus, cytomegalovirus (CMV),varicella zoster virus (VZV), rhinovirus, adenovirus, coronavirus,influenza, and parainfluenza; bacterial diseases such as tuberculosis,Mycobacterium avium, and Hansen's disease; infections such as mycosis,chlamydia, Candida, Aspergillus, cryptococcal meningitis, Pneumocystiscarini, cryptosporidiosis, histoplasmosis, toxoplasmosis, malaria,Trypanosoma infection, and leishmaniasis. Examples of the activeingredient of the vaccine for preventing infectious include, but notlimited to, substances derived from microorganisms/pathogens includingbacteria, fungi, protozoa, and viruses which cause infectious diseases,such as antigenic protein, antigen peptide (partial peptide) from saidantigenic protein, polysaccharide, lipid, and a combination thereof or acombination of the substance derived from said microorganisms/pathogensand a carrier.

Examples of the viral antigenic peptide derived from the viral antigeninclude, but are not limited to, influenza matrix protein peptide 58-66(Jager E et al., Int. J. Cancer 67: 54 (1996)), HPV16 E7 peptide 86-93(van Driel W J et al., Eur. J. Cancer 35:946 (1999)), HPV E7 peptide12-20 (Scheibenbogen C et al., J. Immunother 23: 275 (2000)), HPV16 E7peptide 11-20 (Smith J W I et al., J. Clin. Oncol. 21: 1562 (2003)),HSV2 gD (Berman P W et al., Science 227: 1490 (1985)), CMV gB (Frey S Eet al., Infect Dis. 180: 1700 (1999), Gonczol E. et al., Exp. Opin.Biol. Ther. 1: 401 (2001)), and CMV pp 65 (Rosa C L et al., Blood 100:3681 (2002), Gonczol E. et al., Exp. Opin. Biol. Ther. 1: 401 (2001)).

The carrier as used herein is a substance, such as protein and lipid, towhich an antigenic protein or an antigenic peptide is bonded chemicallyand/or physically, and examples include, but are not limited to, CRM 197(Vaccine. 2013 Oct. 1; 31(42):4827-33), KLH (Cancer Immunol Immunother.2003 October; 52(10):608-16), virus-like particles (PLoS ONE 5(3):e9809) and liposomes (J Liposome Res. 2004; 14(3-4):175-89).

The antigenic protein may be prepared by cloning cDNA, which encodes theantigenic protein, and expression in a host cell, according to atextbook such as Molecular Cloning 2nd ed., Cold Spring HarborLaboratory Press (1989).

The synthesis of the antigenic peptide can be carried out according to amethod generally used in peptide chemistry, for example, as described inliteratures (Peptide Synthesis, Interscience, New York, 1966; TheProteins, Vol. 2, Academic Press Inc., New York, 1976).

The present invention further provides a kit comprising:

a) a compound of the formula (1), or a pharmaceutically acceptable saltthereof;

b) an antigen; and

c) a container or device to contain a unit dosage form of a) and b) incombination or separately.

The antigen is not limited so long as it is an antigen that may be usedas an active ingredient of vaccines, and examples include antigenicproteins as mentioned above, antigenic peptides (partial peptides)derived from such antigenic proteins, and a complex thereof with acarrier.

In one embodiment of the present invention, there is provided a use of acompound of the formula (1), or a pharmaceutically acceptable saltthereof, for the manufacture of a vaccine adjuvant.

In one embodiment of the present invention, there is provided a use of acompound of the formula (I) as defined above, or a pharmaceuticallyacceptable salt thereof, as a vaccine adjuvant in the manufacture of avaccine for the treatment of a disease or condition.

One embodiment of the present invention provides a method for thetreatment, prevention of or prevention of the progress of the diseases,comprising a step of administering a compound of the formula (I) asdefined above, or a pharmaceutically acceptable salt thereof, togetherwith an antigen, to a patient.

The present invention will be further described with reference to thefollowing examples which should not be regarded as limiting in anyrespect.

EXAMPLES

THF: tetrahydrofuran

EtOAc: ethyl acetate

NMP: N-methylpyrrolidinone

TEA: triethylamine

The measurement conditions for high performance liquid chromatographymass spectrometry (LCMS) were as follows. The observed MS (m/z) valueswere shown with respect to M+H.

MS detector: LCMS-IT-TOF

HPLC: Shimadzu Nexera X2 LC 30AD

Column: Kinetex 1.7μ C18 100A New column 50×2.1 mm

Flow rate: 1.2 ml/min

Measurement wavelength: 254 nm

Mobile phase: Solution A: 0.1% aqueous formic acid solution

-   -   Solution B: acetonitrile        Time program:        Step time (min)    -   1 0.01-1.40 Solution A: Solution B=90:10 to 5:95    -   2 1.40-1.60 Solution A: Solution B=5:95    -   3 1.61-2.00 Solution A: Solution B=99:1

Example 1(4E,8E,12E,16E,20E)-N-{2-[{4-[(2-amino-4-{[(3S)-1-hydroxyhexan-3-yl]amino}-6-methylpyrimidin-5-yl)methyl]benzyl}(methyl)amino]ethyl}-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenamide

Step 1

Methyl 4-2-(ethoxycarbonyl)-3-oxobutyl)benzoate (3.56 g, 12.8 mmol) andguanidine carbonate (4.61 g, 25.6 mmol) were dissolved in methanol (23ml), and the mixture was heated to reflux with stirring for 7 hour.After cooling the reaction mixture, water (30 ml) and acetic acid (0.660ml, 11.5 mmol) were added. The precipitated solid was collected byfiltration. The solid was suspended in THF and heated to reflux withstirring for one hour. After cooling, the solid was collected byfiltration, washed with THF and dried to obtain the desired product(1.62 g, 46%). ¹H-NMR (400 MHz, DMSO-d⁶) δ2.00 (3H, s), 3.71 (2H, s),3.82 (3H, s), 6.35 (1H, br), 7.31 (2H, d, J=8.3 Hz), 7.84 (2H, d, J=8.3Hz).

Step 2

The compound obtained in Step 1 (1.62 g, 5.93 mmol) andN,N,N′,N′-tetramethyl-1,3-propanediamine (1.41 ml, 8.89 mmol) weresuspended in THF (24 ml), and 2-mesitylenesulfonyl chloride (1.94 g,8.89 mmol) was added. The mixture was stirred at room temperature for 20hours. Water was added to the mixture. The mixture was extracted withethyl acetate. The organic layer was washed with brine, dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. The obtained solid was washed with ether, and with hexane, anddried to obtain the desired product (2.69 g, 99.6%).

¹H-NMR (400 MHz, DMSO-d⁶) δ2.20 (3H, s), 2.29 (3H, s), 2.48 (6H, s),3.84 (3H, s), 3.88 (2H, s), 6.35 (1H, br), 7.08 (2H, s), 7.19 (2H, d,J=8.3 Hz), 7.85 (2H, d, J=8.3 Hz).

Step 3

Methyl4-((2-amino-4-((mesitylsulfonyl)oxy)-6-methylpyrimidin-5-yl)methyl)benzoate(3.6 g, 8.0 mmol) was dissolved in propionitrile (80 ml).(S)-(+)-3-amino-1-hexanol (5.6 g, 48 mmol) and trifluoroacetic acid (1.2ml, 16 mmol) were added, and the mixture was heated to 110° C. Afterstirring for 36 hours, the mixture was cooled to room temperature andconcentrated under reduced pressure. The residue was purified on silicagel column (ethyl acetate:methanol=20:1 to 5:1) to obtain the desiredproduct (2.1 g, 71%).

¹H-NMR (CDCl₃) δ 0.73 (t, J=7.2 Hz, 3H), 0.90-1.76 (m, 6H), 2.41 (s,3H), 3.49-3.61 (m, 2H), 3.69-3.87 (m, 2H), 3.90 (s, 3H), 4.24 (m, 1H),7.17 (d, J=8.0 Hz, 2H), 7.98 (d, J=8.0 Hz, 2H).

Step 4

(S)-Methyl4-((2-amino-4-((1-hydroxyhexan-3-yl)amino)-6-methylpyrimidin-5-yl)methyl)benzoate](2.1 g, 5.6 mmol) was dissolved in tetrahydrofuran (56 mL)/methanol (5.6mL). Lithium borohydride (3M in tetrahydrofuran, 5.6 mL, 17 mmol) wasadded, and the mixture was heated to 60° C. After stirring for 2 hours,lithium borohydride (3M in tetrahydrofuran, 5.6 mL, 17 mmol) was added,and the mixture was further stirred at 60° C. for 2 hours. After coolingto 0° C., 4N hydrochloric acid (30 mL) was added. The mixture wasstirred at room temperature for 1 hour, neutralized with aqueous sodiumhydrogen carbonate solution, and then extracted with ethyl acetate. Theorganic layer was washed with brine, dried over magnesium sulfate, andconcentrated under reduced pressure to obtain a crude product (1.2 g).

¹H-NMR (CD₃OD) δ 0.78 (t, J=7.2 Hz, 3H), 1.03-1.76 (m, 6H), 2.18 (s,3H), 3.41-3.48 (m, 2H), 3.76-3.88 (m, 2H), 4.23 (m, 1H), 4.55 (s, 2H),7.11 (d, J=8.0 Hz, 2H), 7.26 (d, J=8.0 Hz, 2H).

Step 5

(S)-3-((2-Amino-5-(4-(hydroxymethyl)benzyl)-6-methylpyrimidin-4-yl)amino)hexan-1-ol(1.2 g, 3.5 mmol) was dissolved in chloroform (35 mL)/methanol (3.5 mL),and manganese dioxide (3.1 g, 35 mmol) was added. The mixture wasstirred overnight and filtered through celite. The filtrate wasconcentrated under reduced pressure. The residue was purified on silicagel column (chloroform:methanol=99:1 to 4:1) to obtain the desiredproduct (0.72 g, 37% in 2 Steps).

¹H-NMR (CD₃OD) δ 0.70 (t, J=7.2 Hz, 3H), 1.05-1.77 (m, 6H), 2.20 (s,3H), 3.42-3.50 (m, 2H), 3.90-4.03 (m, 2H), 4.32 (m, 1H), 7.35 (d, J=8.0Hz, 2H), 7.84 (d, J=8.0 Hz, 2H), 9.93 (s, 1H).

Step 6

(S)-4-((2-Amino-4-((1-hydroxyhexan-3-yl)amino)-6-methylpyrimidin-5-yl)methyl)benzaldehyde(0.28 g, 0.82 mmol) was dissolved in chloroform (8 mL). t-Butyl(2-(methylamino)ethyl)carbamate (0.29 g, 1.6 mmol), acetic acid (0.23mL, 4.1 mmol) and sodium triacetoxyborohydride (0.52 g, 2.5 mmol) wereadded. The mixture was stirred overnight at room temperature. Water wasadded, and the mixture was extracted with ethyl acetate. The organiclayer was washed with brine, dried over magnesium sulfate, concentratedunder reduced pressure. The residue was purified on amino silica gelcolumn (ethyl acetate:methanol=99:1 to 95:5) to obtain a crude product(0.23 g, 56%).

¹H-NMR (CDCl₃) δ 0.66 (t, J=7.2 Hz, 3H), 0.91-1.77 (m, 6H), 1.40 (s,9H), 2.12 (s, 3H), 2.26 (s, 3H), 2.39-2.44 (m, 2H), 3.15-3.46 (m, 4H),3.43 (s, 2H), 3.61-3.86 (m, 2H), 4.05 (m, 1H), 4.67-4.77 (br, 2H),4.91-5.01 (br, 1H), 7.05 (d, J=8.0 Hz, 2H), 7.18 (d, J=8.0 Hz, 2H).

Step 7

(S)-Butyl(2-((4-((2-amino-4-((1-hydroxyhexan-3-yl)amino)-6-methylpyrimidin-5-yl)methyl)benzyl)(methyl)amino)ethyl)carbamate (0.150 g, 0.29 mmol) was dissolved inchloroform (4 mL). Hydrogen chloride (4M in cyclopentyl methyl ether,2.2 ml, 8.80 mmol) was added. The mixture was stirred at roomtemperature for 1 hour and then concentrated under reduced pressure.(4E,8E,12E,16E,20E)-4,8,12,17,21,25-Hexamethylhexacosa-4,8,12,16,20,24-hexaenoicacid (123 mg, 0.262 mmol),1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridin-1-ium3-oxide hexafluorophosphate (150 mg, 0.393 mmol) andN,N-diisopropylethylamine (0.137 ml, 0.787 mmol) were dissolved inN,N-dimethyl formamide (3 ml), and the mixture was stirred for 5minutes. The crude product was dissolved in N,N-dimethyl formamide (3ml) and added to the mixture. The mixture was stirred overnight at roomtemperature. Water was added, and the mixture was extracted with ethylacetate. The organic layer was washed with brine, dried over magnesiumsulfate, and concentrated under reduced pressure. The residue waspurified on amino silica gel column (chloroform:methanol=99:1 to 90:10)to obtain the desired product (80 mg, 32%).

¹H-NMR (CDCl₃) δ 0.71 (t, J=7.2 Hz, 3H), 0.92-1.83 (m, 27H), 1.95-2.08(m, 20H), 2.18-2.32 (m, 10H), 2.46 (t, J=6.0 Hz, 2H), 3.28-3.43 (m, 4H),3.46 (s, 2H), 3.64-3.81 (m, 2H), 4.10 (m, 1H), 4.48-4.58 (br, 2H),5.06-5.17 (m, 6H), 5.94-6.04 (br, 1H), 7.06 (d, J=8.0 Hz, 2H), 7.20 (d,J=8.0 Hz, 2H). ESI: [M+H]⁺ 852.6

Example 2(4E,8E,12E,16E,20E)-1-(4-{4-[(2-amino-4-{[(2S)-1-hydroxypentan-2-yl]amino}-6-methylpyrimidin-5-yl)methyl]-3-methoxybenzyl}piperazin-1-yl)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaen-1-one

The desired product (96 mg, 23% in 2 Steps) was obtained in the similarmanner as Step 6 and Step 7 of Example 1, using known compound(S)-4-((2-amino-4-((1-hydroxypentan-2-yl)amino)-6-methylpyrimidin-5-yl)methyl)-3-methoxybenzaldehyde(0.10 g, 0.28 mmol), t-butyl piperazin-1-carboxylate (0.13 g, 0.71 mmol)and(4E,8E,12E,16E,20E)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenoicacid (72 mg, 0.15 mmol).

¹H-NMR (CDCl₃) δ 0.77 (t, J=7.2 Hz, 3H), 1.00-1.44 (m, 4H), 1.57-1.65(m, 21H), 1.88-2.04 (m, 20H), 2.23-2.38 (m, 11H), 3.38-3.74 (m, 6H),3.44 (s, 2H), 3.67 (s, 2H), 3.88 (s, 3H), 4.00 (m, 1H), 4.77-4.87 (br,2H), 5.03-5.11 (m, 6H), 6.79 (d, J=8.0 Hz, 1H), 6.86 (s, 1H), 6.89 (d,J=8.0 Hz, 1H). ESI: [M+H]⁺ 879.6

Example 3(4E,8E,12E,16E,20E)-1-(4-{4-[(2-amino-4-{[(2S)-1-hydroxypentan-2-yl]amino}-6-methylpyrimidin-5-yl)methyl]-3-methoxybenzoyl}piperazin-1-yl)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaen-1-one

Step 1

(S)-4-((2-Amino-4-((l-hydroxypentan-2-yl)amino)-6-methylpyrimidin-5-yl)methyl)-3-methoxybenzoicacid (190 mg, 0.507 mmol), which was prepared as described inWO2012/066336, and 1-BOC-piperazine (142 mg, 0.761 mmol) were dissolvedin N,N-diethylformamide (5 ml).1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (289 mg, 0.761 mmol) andN,N-diisopropylethylamine (197 mg, 1.52 mmol) were added, and themixture was stirred at room temperature for 10 hours. Water was added tothe mixture, and the mixture was extracted with chloroform. The organiclayer was dried over anhydrous sodium sulfate, filtered, andconcentrated under reduced pressure. The resulting residue was purifiedon silica gel column chromatography (ethyl acetate/methanol=20/1 to 5/1)to obtain the desired product (202 mg, 73%) as colorless amorphous. MS(ESI+): [M+H]⁺ 543.4

Step 2

(S)-tert-Butyl4-(4-((2-amino-4-((1-hydroxypentan-2-yl)amino)-6-methylpyrimidin-5-yl)methyl)-3-methoxybenzoyl)piperazin-1-carboxylate(200 mg, 0.369 mmol) was dissolved in chloroform (3.0 ml). 4MHydrochloric acid-cyclopentyl methyl ether solution (3 ml, 12.0 mmol)was added, and the mixture was stirred at room temperature for 1 hour.Toluene was added to the reaction mixture, and the mixture wasconcentrated under reduced pressure. The residue was added to thesolution of(4E,8E,12E,16E,20E)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenoicacid (166 mg, 0.355 mmol),1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (202 mg, 0.532 mmol) and N,N-diisopropylethyl amine (0.185 ml, 1.065 mmol) in N,N-dimethyl formamide (5 ml), andthe mixture was stirred at room temperature for 6 hours. Water was addedto the mixture, and the mixture was extracted with chloroform. Theorganic layer was washed with brine, dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure. The residuewas purified on silica gel column chromatography (chloroform/methanol20/1) to obtain the title compound (159 mg, 50%).

¹H-NMR (CDCl₃) δ 0.83 (t, J=7.3 Hz, 3H), 1.15-1.48 (m, 4H), 1.58-1.68(m, 21H), 1.94-2.15 (m, 20H), 2.25-2.32 (m, 2H), 2.32 (s, 3H), 2.40-2.48(m, 2H), 3.37-3.80 (m, 12H), 3.93 (s, 3H), 3.99-4.05 (m, 1H), 4.65 (brs,2H), 4.80 (br, 1H), 5.07-5.18 (m, 6H), 6.86 (dd, J=7.8, 1.4 Hz, 1H),6.97-7.00 (m, 2H). MS (ESI+): [M+H]⁺ 893.6

Example 44-[(2-amino-4-{[(2S)-1-hydroxypentan-2-yl]amino}-6-methylpyrimidin-5-yl)methyl]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenoyl]amino}ethyl)-3-methoxybenzamide

Step 1

The title compound (216 mg, 78%) was obtained, in the similar manner asStep 1 of Example 3, using(5)-4-((2-amino-4-((1-hydroxypentan-2-yl)amino)-6-methylpyrimidin-5-yl)methyl)-3-methoxybenzoicacid (0.14 g, 0.41 mmol), which was prepared as described inWO2012/066336, and tert-butyl (2-aminoethyl)carbamate (137 mg, 0.855mmol).

¹H-NMR (CDCl₃) 5 0.81 (t, J=7.3 Hz, 3H), 1.07-1.40 (m, 4H), 1.47 (s,9H), 2.32 (s, 3H), 3.37-3.45 (m, 3H), 3.52-3.57 (m, 2H), 3.63-3.67 (m,1H), 3.74 (s, 2H), 3.97 (s, 3H), 3.97-4.03 (m, 1H), 4.58 (br, 2H),4.75-4.77 (br, 1H), 4.97-4.99 (br, 1H), 6.95-6.98 (m, 1H), 7.22-7.23 (m,1H), 7.27-7.31 (br, 1H), 7.47-7.49 (m, 1H).

Step 2

The title compound (84.0 mg, 24%) was obtained, in the similar manner asStep 2 of Example 3, using the compound obtained in Step 1 (205 mg,0.397 mmol).

¹H-NMR (CDCl₃) δ 0.81 (t, J=7.3 Hz, 3H), 1.07-1.46 (m, 4H), 1.55-1.70(m, 21H), 1.90-2.10 (m, 20H), 2.26-2.30 (m, 4H), 2.33 (s, 3H), 3.38-3.44(m, 1H), 3.46-3.58 (m, 4H), 3.62-3.67 (m, 1H), 3.74 (s, 2H), 3.97 (s,3H), 3.97-4.03 (m, 1H), 4.54-4.57 (br, 2H), 4.76-4.79 (br, 1H),5.06-5.15 (m, 6H), 6.10-6.14 (br, 1H), 7.00 (d, J=8.0 Hz, 1H), 7.24 (dd,J=8.0, 1.6 Hz, 1H), 7.38-7.41 (br, 1H), 7.45 (d, J=1.6 Hz, 1H). MS(ESI+): [M+H]⁺ 867.6.

Example 54-[(2-amino-4-{[(2S)-1-hydroxypentan-2-yl]amino}-6-methylpyrimidin-5-yl)methyl]-N-(2-{[(4E,8E,12E,16E,20E)-4,8,12,17,21,25-hexamethylhexacosa-4,8,12,16,20,24-hexaenoyl](methyl)amino}ethyl)-3-methoxybenzamide

Step 1

The title compound (242 mg, 81%) was obtained, in the similar manner asStep 1 of Example 3, using(5)-4-((2-amino-4-((1-hydroxypentan-2-yl)amino)-6-methylpyrimidin-5-yl)methyl)-3-methoxybenzoicacid (0.14 g, 0.41 mmol), which was prepared as described inWO2012/066336, and N-(2-aminoethyl)-N-methyl carbamic acid t-butyl ester(98 mg, 0.561 mmol).

¹H-NMR (CDCl₃) δ 0.84 (t, J=7.3 Hz, 3H), 1.07-1.41 (m, 4H), 1.43 (s,9H), 2.31 (s, 3H), 2.91 (s, 3H), 3.39-3.44 (m, 1H), 3.45-3.55 (m, 2H),3.56-3.61 (m, 2H), 3.62-3.67 (m, 1H), 3.73 (s, 2H), 3.96 (s, 3H),3.97-4.02 (m, 1H), 4.55-4.59 (br, 2H), 4.74-4.78 (br, 1H), 6.94-6.97 (m,1H), 7.21-7.24 (m, 1H), 7.47-7.50 (m, 2H).

Step 2

The title compound (149 mg, 38%) was obtained, in the similar manner asStep 2 of Example 3, using the compound obtained in Step 1 (236 mg,0.445 mmol).

¹H-NMR (CDCl₃) δ 0.80 (t, J=7.3 Hz, 3H), 1.06-1.45 (m, 4H), 1.56-1.68(m, 21H), 1.90-2.09 (m, 20H), 2.21-2.31 (m, 2H), 2.33 (s, 3H), 2.37-2.43(m, 2H), 3.08 (s, 3H), 3.37-3.45 (m, 1H), 3.55-3.69 (m, 5H), 3.73 (s,2H), 3.96 (s, 3H), 3.97-4.03 (m, 1H), 4.55-4.58 (br, 2H), 4.77-4.80 (br,1H), 5.06-5.14 (m, 6H), 7.00 (d, J=8.0 Hz, 1H), 7.24 (dd, J=8.0, 1.2 Hz,1H), 7.44 (d, J=1.6 Hz, 1H), 7.53-7.55 (br, 1H). MS (ESI+): [M+H]⁺ 881.6

Test Example 1

For the first immunization, an equal volume mixture (100 μL/mouse) ofovalbumin (OVA) (1 mg/mL) and a compound of the above Examples (0.1mg/mL) was administered intramuscularly to gastrocnemius muscle ofC57BL/6 mouse. Two weeks later, an equal amount of the same mixture wasadministered intramuscularly to the gastrocnemius muscle for boosterimmunization. One week after the booster immunization, cardiac blood wascollected under isoflurane inhalation anesthesia, and plasma wascollected by centrifugation. OVA-specific IgG value in plasma wasmeasured by ELISA using mouse anti-ovalbumin IgG ELISA kit (AlphaDiagnostic) (See FIG. 1).

Also, the OVA-specific IgG1 and IgG2c values in the immunized mouseplasma were determined by ELISA method. Specifically, OVA solution(SIGMA) was added to a 96-well plate, followed by blocking with 1% skimmilk (Wako Pure Chemical Industries, Ltd.). A plasma sample, which wasdiluted with phosphate buffer, and then a secondary antibody (goatanti-mouse IgG1 (Jackson) or IgG2c (Southern Bio)) were added. SureBlue™TMB microwell peroxidase substrate (KPL) was added, and the product ofthe enzyme reaction was measured by microplate reader (See FIG. 2).

All of the compounds of Examples 1 to 5 induced OVA-specific IgG morestrongly as compared with the negative control group. In particular, thecompounds of Example 1 and Example 4 induced significant OVA-specificIgG production, as compared with the negative control group.Furthermore, the compounds of Example 1 and Example 4 significantlyincreased the ratio of IgG2c (one of Th1-type IgGs) to IgG1 (one ofTh2-type IgGs), as compared with the negative control group.

Test Example 2

The spleen cells were prepared from the mouse of Test Example 1. Thecells were added with OVA and Brefeldin A (eBioscience) and culturedovernight. The cultured cells were harvested, stained with APC-labeledanti-mouse CD3e antibody, FITC-labeled anti-mouse CD4 antibody andFixable Viability Dye Fluor® 450 (eBioscience), and fixed inFixation/Permeabilization buffer (eBioscience). After treatment inPermeabilization buffer (eBioscience), the cells were stained withantibody cocktail PerCP-Cy5.5-labeled anti-IFN-γ antibody,PE-Cy7-labeled anti-IL-2 antibody, and PE-labeled TNF-α (eBioscience).Data acquisition and analysis were performed using FACS Cant II (BDBiosciences) and FLOWJO software (TreeStar). The results were shown inFIG. 3.

The spleen cells were also stained with eFluor 450-labeled anti-mouseCD3e antibody, Alexa Fluor® 647-labeled anti-mouse CD8 antibody,PE-labeled H-2K^(b) OVA Tetramer-SINFEKL (MBL) and Flexable ViabilityDye eFluor 520 (eBioscience). Data acquisition and analysis wereperformed using FACS Cant II (BD Biosciences) and FLOWJO software(TreeStar). The results are shown in FIG. 4.

The spleen cells were further incubated with antibody cocktaileFluor450-labeled anti-mouse CD3e antibody, Alexa Fluor647-labeledanti-mouse CD8 antibody, PE-Cy7-labeled anti-mouse CD44 antibody,PerCP-Cy5.5-labeled anti-mouse CD62L antibody and Fixable ViabilityDye520 (eBioscience). Data acquisition and analysis were performed usingFACS Cant II (BD Biosciences) and FLOWJO software (TreeStar). Theresults were shown in FIG. 5.

The compounds of Example 1 and Example 4 significantly increased theproportion of OVA-specific multifunctional CD4-positive T lymphocytes,the proportion of MHC-restricted OVA-specific CD8-positive Tlymphocytes, and the proportion of CD8-positive effector memory Tlymphocytes, as compared with the negative control group.

INDUSTRIAL APPLICABILITY

The compound of the present invention is useful as an adjuvant, which isadded to a vaccine preparation for enhancement of the immunostimulatingeffect.

The invention claimed is:
 1. A compound of the formula (2):

or a pharmaceutically acceptable salt thereof, wherein: X is —CH₂; Y¹ is—(CR⁹R¹⁰)_(p)—; Y² is a single bond; L is a straight chain C₂₋₆alkylene, wherein the straight chain C₂₋₆ alkylene is optionallysubstituted with 1, 2, 3, or 4 substituents independently selected fromthe group consisting of halogen and hydroxy; R¹ is hydrogen or C₁₋₆alkyl, wherein the C₁₋₆ alkyl is optionally substituted with 1, 2, 3, or4 substituents independently selected from the group consisting ofhydroxy halogen, and C₁₋₆ alkoxy; R² is hydrogen or C₁₋₆ alkyl, whereinthe C₁₋₆ alkyl is optionally substituted with 1, 2, 3, or 4 substituentsindependently selected from the group consisting of halogen, hydroxy,and C₁₋₆ alkoxy; R³ is C₁₋₆ alkyl; R⁴ is hydrogen, halogen, cyano, C₁₋₆alkyl, hydroxy, or C₁₋₆ alkoxy; R⁵ is hydrogen or C₁₋₆ alkyl, whereinthe C₁₋₆ alkyl is optionally substituted with 1, 2, 3, or 4 substituentsindependently selected from the group consisting of hydroxy and halogen;R⁶ is hydrogen or C₁₋₆ alkyl, wherein the C₁₋₆ alkyl is optionallysubstituted with 1, 2, 3, or 4 substituents independently selected fromthe group consisting of halogen and hydroxy; R⁸ is hydrogen orC(O)OC(CH₃)₃; R⁹ is hydrogen or C₁₋₄ alkyl; R¹⁰ is hydrogen or C₁₋₄alkyl; and p is 1, 2, 3, 4, 5, or
 6. 2. The compound according to claim1, or a pharmaceutically acceptable salt thereof, wherein: Y¹ is —CH₂—;L is a straight chain C₂₋₃ alkylene, R¹ is hydrogen or C₁₋₃ alkyl,wherein the C₁₋₃ alkyl is optionally substituted with 1, 2, 3, or 4hydroxy substituents; R² is hydrogen or C₁₋₆ alkyl; R³ is C₁₋₃ alkyl; R⁴is hydrogen, halogen, C₁₋₃ alkyl, hydroxy, or C₁₋₃ alkoxy; R⁵ ishydrogen or C₁₋₃ alkyl; and R⁶ is hydrogen or C₁₋₃ alkyl.
 3. Thecompound according to claim 1, wherein the compound is selected from thegroup consisting of:

or a pharmaceutically acceptable salt thereof.
 4. The compound accordingto claim 3, wherein the compound is:

or a pharmaceutically acceptable salt thereof.
 5. The compound accordingto claim 3, wherein the compound is:

or a pharmaceutically acceptable salt thereof.